Antifreeze proteins (AFPs) are natural antifreeze molecules that have been found in many organisms including fish, insects, plants, bacteria, and fungi. AFPs exhibit great structural diversity, while they are all characterized by their unique ability to depress the freezing point of water without affecting the melting point apparently. The resulting difference between the melting point and the freezing point, referred to as thermal hysteresis (TH), is generally used as a measure of the antifreeze activities of AFPs. AFPs and co-solutes in body-fluids of cold-adapted organisms are responsible for survival in cold environments. Certain small molecule and macromolecule co-solutes can further enhance the antifreeze activity of AFPs, referred to as enhancers, and they usually do not have TH activity, but can bind to AFP through ionic interactions, hydrogen bonding, and/or hydrophobic interactions. AFP-based antifreeze systems (AFP plus enhancer) are much more effective and their uses are more environmentally friendly in comparison to conventional antifreezes, making them intriguing alternatives to conventional antifreezes in particular in biomedical fields. The proposed research will use series of beetle AFP-small molecule enhancer systems, in which small molecule enhancers display a wide range of antifreeze enhancement abilities. We will determine the binding characteristics between AFPs and enhancers using chemical and biophysical approaches in these systems and unravel the AFP-enhancer affinity in highly efficient AFP systems. We will also determine the factors that can affect the binding in the AFP-enhancers systems. The salt effects will be determined. We will optimize these factors in the proposed functional examinations of the AFP-enhancer systems. Quantitative understanding AFP-based antifreeze systems would help unravel survival strategy in cold-adapted organisms and lead to rational development of highly efficient cold preservation, antifreeze systems for practical biomedical applications, such as in preservation solutions for organ, tissue and cell transplantation, cryopreservation, and cryosurgery.